Different vitamin B12 and folic acid concentrations could exacerbate the immune response. The aim was to evaluate different dietary folic acid and vitamin B12 levels on the immune response in aged rats. Male Sprague Dawley aged rats were assigned to three folic acid groups (deficient, control, supplemented) each in absence of vitamin B12 for 30 days. Several parameters of innate and acquired immune responses were measured. Serum and hepatic folate levels increased according to folic acid dietary level, while vitamin B12 levels decreased. There was a significant decrease in natural killer cell-mediated cytotoxicity in the spleen for the vitamin B12 deficient diet and folic acid control diet groups. Significant changes in CD45 lymphocyte subsets were also observed according to dietary imbalance. Lymphoproliferative response to concanavalin A and phytohemagglutinin did not differ significantly between groups. The spleen response to lipopolysaccharide increased significantly, but was unmodified for the other organs. An imbalance between dietary vitamin B12 and folic acid concentrations alters some immunological parameters in aged rats. Therefore, the ratio between folate and vitamin B12 could be as important as their absolute dietary concentrations.